Long-term demographic and genetic effects of releasing captive-born individuals into the wild

Because of continued habitat destruction and species extirpations, the need to use captive breeding for conservation purposes has been increasing steadily. However, the long-term demographic and genetic effects associated with releasing captive-born individuals with varied life histories into the wild remain largely unknown. To address this question, we developed forward-time, agent-based models for 4 species with long-running captive-breeding and release programs: coho salmon (Oncorhynchus kisutch), golden lion tamarin (Leontopithecus rosalia), western toad (Anaxyrus boreas), and Whooping Crane (Grus americana). We measured the effects of supplementation by comparing population size and neutral genetic diversity in supplemented populations to the same characteristics in unaltered populations 100 years after supplementation ended. Releasing even slightly less fit captive-born individuals to supplement wild populations typically resulted in reductions in population sizes and genetic diversity over the long term when the fitness reductions were heritable (i.e., due to genetic adaptation to captivity) and populations continued to be regulated by density-dependent mechanisms over time. Negative effects for species with longer life spans and lower rates of population replacement were smaller than for species with shorter life spans and higher rates of population replacement. Programs that released captive-born individuals over fewer years or that avoided breeding individuals with captive ancestry had smaller reductions in population size and genetic diversity over the long term. Relying on selection in the wild to remove individuals with reduced fitness mitigated some negative demographic effects, but at a substantial cost to neutral genetic diversity. Our results suggest that conservation-focused captive-breeding programs should take measures to prevent even small amounts of genetic adaptation to captivity, quantitatively determine the minimum number of captive-born individuals to release each year, and fully account for the interactions among genetic adaptation to captivity, population regulation, and life-history variation.     

Population Persistence and Offspring Fitness in the Rare Bellflower Campanula cervicaria in Relation to Population Size and Habitat Quality

Abstract: Data from several animal species and a few plant species indicate that small populations face an elevated risk of extinction. Plants are still underrepresented in these studies concerning the relation between population size and persistence. We studied the effect of population size on persistence among natural populations of the rare bellflower Campanula cervicaria in Finland. We monitored 52 bellflower populations for 8 years and found that the mean population size decreased from 24 to 14 during this period. Small populations with ≤5 individuals were more prone to losing all fertile plants than were larger ones. Reduction in population size was nevertheless unrelated to the degree of population isolation, measured as the distance to the nearest known population. To test the hypothesis that offspring fitness is lower in small populations, we germinated bellflower seeds from different-sized populations in a laboratory and found that seed germination ability was independent of population size. The seedlings raised from seeds of small populations grew faster than those taken from larger populations. Population size was negatively related to the amount of shade in the habitats. In conclusion, decreasing population sizes of C. cervicaria seemed not to be caused by lowered germination ability or growth rate in small populations; rather, population size reductions appeared to be due to closing of vegetation in the habitats.     

Conservation of Species in Dynamic Landscapes: Divergent Fates of Silene tatarica Populations in Riparian Habitats

Abstract:  In transient environments, where local extinctions occur as a result of destruction or deterioration of the local habitat, the long-term persistence of a species requires successful colonizations at new, suitable sites. This kind of habitat tracking should be associated with the asynchronous dynamics of local populations, and it can be especially important for the conservation of rare plant species in riparian habitats. We determined spatiotemporal variation in the demography of the perennial Silene tatarica (L.) Pers. in 15 populations (1998–2003) located in periodically disturbed riparian habitats. The habitats differed according to their morphology (flat shores, slopes) and the amount of bare ground (open, intermediate, closed) along a successional gradient. We used elasticity and life-table response analyses and stochastic simulations to study the variation in population demography. Finite population growth rate was higher in intermediate habitats than in open and closed habitats. In stochastic simulations population size increased in most cases, but four populations were projected to become extinct within 12–70 years. The viability of local populations depended most on the survival and growth of juvenile individuals and on the fecundity of large fertile individuals. On a regional scale, the persistence of this species will require a viable network of local populations as protection against local extinctions caused by natural disturbances and succession. Accordingly, the long-term persistence of riparian species may depend on habitat changes; thus, their conservation requires maintenance of natural disturbance dynamics. Along regulated rivers, management activities such as the creation of open habitats for new colonization should be implemented. Similarly, these activities can be rather general requirements for the conservation of endangered species dependent on transient habitats along successional gradients.     

Mutation and Conservation

Mutation can critically affect the viability of small populations by causing inbreeding depression, by maintaining potentially adaptive genetic variation in quantitative characters, and through the erosion of fitness by accumulation of mildly detrimental mutations. I review and integrate recent empirical and theoretical work on spontaneous mutation and its role in population viability and conservation planning. I analyze both the maintenance of potentially adaptive genetic variation in quantitative characters and the role of detrimental mutations in increasing the extinction risk of small populations. Recent experiments indicate that the rate of production of quasineutral, potentially adaptive genetic variance in quantitative characters is an order of magnitude smaller than the total mutational variance because mutations with large phenotypic effects tend to be strongly detrimental. This implies that, to maintain normal adaptive potential in quantitative characters under a balance between mutation and random genetic drift (or among mutation, drift, and stabilizing natural selection), the effective population size should be about 5000 rather than 500 (the Franklin-Soulé number). Recent theoretical results suggest that the risk of extinction due to the fixation of mildly detrimental mutations may be comparable in importance to environmental stochasticity and could substantially decrease the long-term viability of populations with effective sizes as large as a few thousand. These findings suggest that current recovery goals for many threatened and endangered species are inadequate to ensure long-term population viability.     

The inflationary effects of environmental fluctuations ensure the persistence of sink metapopulations

Under current rates of environmental change many populations may be found in habitats of low quality and low conservation value, creating population sinks. We test recent theory that suggests, surprisingly, that stochastic environmental variability may enhance the long-term persistence of sink metapopulations. Using experimental populations of Paramecium aurelia we show that it is possible for a metapopulation comprised entirely of sink populations to persist for many generations in a random environment. In accordance with the theory, we show that positive temporal autocorrelation and low spatial correlation in the environment can ensure the long-term persistence and enhance the mean and maximum abundance of sink metapopulations. High levels of spatial correlation in the environment created strong population synchrony and limited the persistence time of the sink metapopulations. These results have important implications for the development of a theory underlying the synergistic effects of habitat fragmentation and environmental change on population persistence.     

Population feedback after successful invasion leads to ecological suicide in seasonal environments

For most consumer species, winter represents a period of harsh food conditions in addition to the physiological strain that results from the low ambient temperatures. In size-structured populations, larger-bodied individuals do better during winter as they have larger energy reserves to buffer starvation periods. In contrast, smaller-bodied individuals do better under growing conditions, as they have lower maintenance costs. We study how the interplay between size-dependent life-history processes and seasonal changes in temperature and food availability shape the long-term dynamics of a size-structured consumer population and its unstructured resource. We show that the size dependence of maintenance requirements translates into a minimum body size that is needed for surviving starvation when consumers can adapt only to a limited extent to the low food densities in winter. This size threshold can lead to population extinction because adult individuals suffer only a little during winter and hence produce large numbers of offspring. Due to population feedback on the resource and intense intra-cohort competition, newborn consumers then fail to reach the size threshold for survival. Under these conditions, small numbers of individuals can survive, increase in density, and build up a population, which will subsequently go extinct due to its feedback on the resource. High juvenile mortality may prevent this ecological suicide from occurring, as it releases resource competition among newborns and speeds up their growth. In size-structured populations, annual fluctuations in temperature and food availability may thus lead to a conflict between individual fitness and population persistence.     

Experimental Tests of Captive Breeding for Endangered Species

Abstract: Several captive breeding regimes were compared for their ability to maintain fitness ( larval viability) and genetic variation in small populations of the housefly ( Musca domestica L.). Populations were either maintained at constant sizes of 40, 200, or 2000 individuals or initiated with two pairs of flies and allowed to grow to 40 individuals ( low-founder-number populations). Low-founder-number populations without migration exhibited low larval viability (22%) after 24 generations, compared to larger populations maintained at either 200 (49%) or 2000 (69%) individuals, and suffered high extinction, with only 44% of the lines surviving 24 generations. Low-founder-number populations subjected to two additional founder ( bottleneck) episodes, reducing them to two pairs of flies, suffered little additional loss in fitness or extinction compared to the single-founder treatments. Migration as low as one individual per generation (2.5% migration) significantly offset both reduced fitness and rate of extinction. Conversely, fitness was not significantly increased for low-founder-number populations when founders were selected from the top performing 20% of pairs under full-sib mating. Populations maintained at 40 individuals were not sustainable, exhibiting low larval viability (35%) and a high extinction rate (40%) over 24 generations, similar to the extinction rates for populations initiated with only four founders. Although none of the populations maintained at 200 individuals went extinct, their fitness was reduced by 20% compared to a large control population maintained at 2000 individuals. Electrophoretic variation was significantly correlated with fitness across treatments, but the correlation of fitness to narrow-sense heritability of two morphometric traits was not significant.     

Minimise long-term loss or maximise short-term gain?: Optimal translocation strategies for threatened species

Translocation is a useful management option for conservation of threatened animal species. It can be used to increase the range of a species, augment the numbers in a critical population, or establish new populations and hence spread the risk of extinction through local catastrophes. As it is an important and expensive conservation tool, translocation management decisions must be carefully considered, with the objective of the translocation project in mind. By analysing the translocation problem within a decision-theory framework, we find optimal management decisions that are rational and transparent. We illustrate our approach using a case study of the bridled nailtail wallaby (Onychogalea fraenata). Our particular translocation question is: if we have a set number of wallabies to translocate in each time period and two translocation sites, how many wallabies should we put at each site given the state of each population to maximise the benefit to the species? We model the translocated populations with first-order Markov chain stochastic population models, and use stochastic dynamic programming to determine the optimal management decisions. We look at two sites with different growth rates – one increasing and one decreasing – and compare the optimal strategies for two different objective functions. The first is a long-term persistence objective function, which maximises the persistence of translocated populations a large number of time steps after the end of the translocation program. The second maximises total population size at the end of the translocation program. Although these objective functions are similar, they generate surprisingly different optimal translocation strategies. When maximising the long-term persistence of the translocated populations, translocation decisions are not important as long as an increasing population is established. This indicates that site quality – rather than the number and timing of translocations – primarily determines the long-term persistence of populations. When maximising total population size, the optimal strategy is to add to the increasing population unless it is above a size where it is likely to reach its carrying capacity over the planning timeframe. As translocation decisions are important in fulfilling the objective, this objective function is more useful in creating practical advice for translocation managers. The discrepancy between the optimal strategies given by the two objectives demonstrates the importance of careful consideration when specifying the goals of a project. This observation applies not only to translocation programs, but any project where clear decision-making is needed.     

Conservation Implications of the Natural Loss of Lineages in Wild Mammals and Birds

Because populations in zoological parks and nature reserves often are derived from only a few individuals, conservationists have attempted to minimize founder effects by equalizing family group sizes and increasing the reproductive contributions of all individuals. Although such programs reduce potential losses of genetic diversity, information is rarely available about the actual persistence of family groups or genetic lineages in natural populations. In the absence of such data, it can be difficult to weigh the importance of human intervention in the conservation of small populations. Separate long-term studies of two mammals, the North American bison (Bison bison) and the white-nosed coati (Nasua narica), and a bird, the Acorn Woodpecker (Melanerpes formicivorus), demonstrate differential extinction of genetic lineages. Irrespective of the mechanisms affecting population structure, which may range from stochastic environmental events to such behavioral phenomena as poor intrasexual competitive abilities, our results show that lineages can be lost at rapid rates from natural populations. A survey of comparable studies from the literature indicates that the loss of matrilines over the course of the study varies from 3% to 87% in wild mammals and from 30% to 80% in birds, with several small mammals losing approximately 20% of matrilines per year of study. These lineage extinctions were not an artifact of the length of the study or the generation time of the species. Such rapid losses of lineages in less than 20-year periods in natural populations suggest that efforts to maintain maximal genetic diversity within populations may not always reflect processes that occur in the wild. Conservation biologists need to give further thought to the extent to which parity among genetic lines should be a primary goal of management of captive and small wild populations.     

Longitudinal monitoring of neutral and adaptive genomic diversity in a reintroduction

Restoration programs in the form of ex-situ breeding combined with reintroductions are becoming critical to counteract demographic declines and species losses. Such programs are increasingly using genetic management to improve conservation outcomes. However, the lack of long-term monitoring of genetic indicators following reintroduction prevents assessments of the trajectory and persistence of reintroduced populations. We carried out an extensive monitoring program in the wild for a threatened small-bodied fish (southern pygmy perch, Nannoperca australis) to assess the long-term genomic effects of its captive breeding and reintroduction. The species was rescued prior to its extirpation from the terminal lakes of Australia's Murray-Darling Basin, and then used for genetically informed captive breeding and reintroductions. Subsequent annual or biannual monitoring of abundance, fitness, and occupancy over a period of 11 years, combined with postreintroduction genetic sampling, revealed survival and recruitment of reintroduced fish. Genomic analyses based on data from the original wild rescued, captive born, and reintroduced cohorts revealed low inbreeding and strong maintenance of neutral and candidate adaptive genomic diversity across multiple generations. An increasing trend in the effective population size of the reintroduced population was consistent with field monitoring data in demonstrating successful re-establishment of the species. This provides a rare empirical example that the adaptive potential of a locally extinct population can be maintained during genetically informed ex-situ conservation breeding and reintroduction into the wild. Strategies to improve biodiversity restoration via ex-situ conservation should include genetic-based captive breeding and longitudinal monitoring of standing genomic variation in reintroduced populations.     

Erosion of Heterozygosity in Fluctuating Populations

Abstract: Demographic, environmental, and genetic stochasticity threaten the persistence of isolated populations. The relative importance of these intertwining factors remains unresolved, but a common view is that random demographic and environmental events will usually drive small populations to the brink of extinction before genetic deterioration poses a serious threat. To evaluate the potential importance of genetic factors, we analyzed a model linking demographic and environmental conditions to the loss of genetic diversity in isolated populations undergoing natural levels of fluctuation. Nongenetic processes—environmental stochasticity and population demography—were modeled according to a bounded diffusion process. Genetic processes were modeled by quantifying the rate of drift according to the effective population size, which was predicted from the same parameters used to describe the nongenetic processes. We combined these models to predict the heterozygosity remaining at the time of extinction, as predicted by the nongenetic portion of the model. Our model predicts that many populations will lose most or all of their neutral genetic diversity before nongenetic random events lead to extinction. Given the abundant evidence for inbreeding depression and recent evidence for elevated extinction rates of inbred populations, our findings suggest that inbreeding may be a greater general threat to population persistence than is generally recognized. Therefore, conservation biologists should not ignore the genetic component of extinction risk when assessing species endangerment and developing recovery plans.     

Role of Patch Size, Disease, and Movement in Rapid Extinction of Bighorn Sheep

Abstract: The controversy (  Berger 1990, 1999 ; Wehausen 1999 ) over rapid extinction in bighorn sheep ( Ovis canadensis ) has focused on population size alone as a correlate to persistence time. We report on the persistence and population performance of 24 translocated populations of bighorn sheep. Persistence in these sheep was strongly correlated with larger patch sizes, greater distance to domestic sheep, higher population growth rates, and migratory movements, as well as to larger population sizes. Persistence was also positively correlated with larger average home-range size ( p = 0.058, n = 10 translocated populations) and home-range size of rams ( p = 0.087, n = 8 translocated populations). Greater home-range size and dispersal rates of bighorn sheep were positively correlated to larger patches. We conclude that patch size and thus habitat carrying capacity, not population size per se, is the primary correlate to both population performance and persistence. Because habitat carrying capacity defines the upper limit to population size, clearly the amount of suitable habitat in a patch is ultimately linked to population size. Larger populations (250+ animals) were more likely to recover rapidly to their pre-epizootic survey number following an epizootic ( p = 0.019), although the proportion of the population dying in the epizootic also influenced the probability of recovery ( p = 0.001). Expensive management efforts to restore or increase bighorn sheep populations should focus on large habitat patches located ≥23 km from domestic sheep, and less effort should be expended on populations in isolated, small patches of habitat.     

Demographic Stability Metrics for Conservation Prioritization of Isolated Populations

Abstract:  Systems of geographically isolated habitat patches house species that occur naturally as small, disjunct populations. Many of these species are of conservation concern, particularly under the interacting influences of isolation and rapid global change. One potential conservation strategy is to prioritize the populations most likely to persist through change and act as sources for future recolonization of less stable localities. We propose an approach to classify long-term population stability (and, presumably, future persistence potential) with composite demographic metrics derived from standard population-genetic data. Stability metrics can be related to simple habitat measures for a straightforward method of classifying localities to inform conservation management. We tested these ideas in a system of isolated desert headwater streams with mitochondrial sequence data from 16 populations of a flightless aquatic insect. Populations exhibited a wide range of stability scores, which were significantly predicted by dry-season aquatic habitat size. This preliminary test suggests strong potential for our proposed method of classifying isolated populations according to persistence potential. The approach is complementary to existing methods for prioritizing local habitats according to diversity patterns and should be tested further in other systems and with additional loci to inform composite demographic stability scores.     

Population Constraints Associated with the Use of Black Rhinos as an Umbrella Species for Desert Herbivores

The numerous tactics used to conserve biodiversity include the designation of protected areas, political change, and research and education, the latter involving paradigms such as insular biogeography and the "umbrella species concept." In Namibia lands removed from national park status in 1970 and currently under the jurisdiction of indigenous people now contain one of the few unfenced populations of black rhinos (Diceros bicornis) remaining in Africa. Theory predicts that the protection of umbrella species will ensure the survival of other biota that require(s) less space. To gauge how well biodiversity might be retained by examining the spatial needs of a small population of black rhinos, I used data gathered under various ecological conditions to estimate mean and minimum population sizes of six large herbivores of the Namib Desert ranging in size from giraffe (Giraffa camelopardalis) to springbok (Antidorcas marsupialis) and ostrich (Struthio camelus). My results indicate that annual differences in rainfall, both within and between seasons, resulted in wide fluctuations in herbivore population sizes for all species except rhino. Although other herbivores switched to areas of higher rainfall, rhinos did not. The data suggest that under conditions of extreme environmental variance the space used by rhinos alone was unlikely to assure the existence of populations of other species in excess of 250 individuals. Fifty percent of the species failed to exceed 150 individuals 50% of the time and one third of the species never attained populations in excess of 50 individuals. However, by employing assumptions about the spatial needs of rhino populations numbering up to 100 individuals, the mean minimum population sizes attained by any of four desert herbivores is 535. A future challenge in using rhinos and other large-bodied species as umbrellas for organisms of either similar or dissimilar trophic levels will be the refinement of estimates of population viability.     

Patterns of Genetic Diversity and Its Loss in Mammalian Populations

Abstract:  Policy aimed at conserving biodiversity has focused on species diversity. Loss of genetic diversity, however, can affect population persistence, evolutionary potential, and individual fitness. Although mammals are a well-studied taxonomic group, a comprehensive assessment of mammalian genetic diversity based on modern molecular markers is lacking. We examined published microsatellite data from populations of 108 mammalian species to evaluate background patterns of genetic variability across taxa and body masses. We tested for loss of genetic diversity at the population level by asking whether populations that experienced demographic threats exhibited lower levels of genetic diversity. We also evaluated the effect of ascertainment bias (a reduction in variability when microsatellite primers are transferred across species) on our assessment of genetic diversity. Heterozygosity did not vary with body mass across species ranging in size from shrews to whales. Differences across taxonomic groupings were noted at the highest level, between populations of marsupial and placental mammals. We documented consistently lower heterozygosity, however, in populations that had experienced demographic threats across a wide range of mammalian species. We also documented a significant ( p = 0.01) reduction in heterozygosity as a result of ascertainment bias. Our results suggest that populations of both rare and common mammals are currently losing genetic diversity and that conservation efforts focused above the population level may fail to protect the breadth of persisting genetic diversity. Conservation policy makers may need to focus their efforts below the species level to stem further losses of genetic resources.     

Emerging prion disease drives host selection in a wildlife population

Infectious diseases are increasingly recognized as an important force driving population dynamics, conservation biology, and natural selection in wildlife populations. Infectious agents have been implicated in the decline of small or endangered populations and may act to constrain population size, distribution, growth rates, or migration patterns. Further, diseases may provide selective pressures that shape the genetic diversity of populations or species. Thus, understanding disease dynamics and selective pressures from pathogens is crucial to understanding population processes, managing wildlife diseases, and conserving biological diversity. There is ample evidence that variation in the prion protein gene (PRNP) impacts host susceptibility to prion diseases. Still, little is known about how genetic differences might influence natural selection within wildlife populations. Here we link genetic variation with differential susceptibility of white-tailed deer to chronic wasting disease (CWD), with implications for fitness and disease-driven genetic selection. We developed a single nucleotide polymorphism (SNP) assay to efficiently genotype deer at the locus of interest (in the 96th codon of the PRNP gene). Then, using a Bayesian modeling approach, we found that the more susceptible genotype had over four times greater risk of CWD infection; and, once infected, deer with the resistant genotype survived 49% longer (8.25 more months). We used these epidemiological parameters in a multi-stage population matrix model to evaluate relative fitness based on genotype-specific population growth rates. The differences in disease infection and mortality rates allowed genetically resistant deer to achieve higher population growth and obtain a long-term fitness advantage, which translated into a selection coefficient of over 1% favoring the CWD-resistant genotype. This selective pressure suggests that the resistant allele could become dominant in the population within an evolutionarily short time frame. Our work provides a rare example of a quantifiable disease-driven selection process in a wildlife population, demonstrating the potential for infectious diseases to alter host populations. This will have direct bearing on the epidemiology, dynamics, and future trends in CWD transmission and spread. Understanding genotype-specific epidemiology will improve predictive models and inform management strategies for CWD-affected cervid populations.     

Is Mutation Accumulation a Threat to the Survival of Endangered Populations?

The accumulation of new deleterious mutations has been predicted to constitute a significant threat to the survival of finite sexually reproducing populations. Three measures of genetic load were made on populations of Drosophila melanogaster maintained at effective population sizes of 25, 50, 100, 250, and 500 for 45 or 50 generations and their outbred base population and a new sample from the same wild population. Genetic loads were measured as fitness differentials between inbred and non-inbred lines derived from each population under both benign ( productivity of single pairs) and competitive (competitive index) conditions. No trend of smaller populations exhibiting greater genetic loads than larger ones was observed under either benign or competitive conditions. Further, genetic loads were similar in captive and wild populations. Frequencies of deleterious and lethal alleles on chromosome II were measured by making the chromosome (approximately 40% of the genome) homozygous using a marked balancer stock. Neither deleterious nor lethal allele frequencies exhibited a relationship with population size. The accumulation of detrimental mutations does not appear to pose a significant threat to finite sexual populations with effective sizes of 25 or more over the 100–200 year time frames considered in most wildlife conservation programs.     

Rapid Extinction of Mountain Sheep Populations Revisited

Abstract: Predicting extinction probabilities for populations of various sizes has been a primary focus of conservation biology. Berger (1990) presented an empirically based extinction model for mountain sheep ( Ovis canadensis ) populations in five southwestern states that predicted disappearance within 50 years of all populations estimated to number 50 sheep or fewer, but essentially no loss in that time period of populations estimated at over 100. The majority of the 122 populations he used in his analysis were from California, but his analysis did not use many of the historical size estimates for these populations. I tested Berger's (1990) model using the complete data set from California and found—contrary to his results—that, for all size classes of population estimates, at least 61% of the populations persisted for 50 years. Also, two predictions from Berger's model were not consistent with the data from California: (1) 10 populations have increased from estimates of 50 or fewer animals to over 100, whereas the Berger model predicted that these populations would only decline to extinction; and (2) of 27 extant populations with long enough records, 85% were estimated at least 50 years ago to be 50 individuals or fewer and should therefore be extinct by now. Berger's model has now failed tests in three states and therefore does not support the strong population size effect on extinction probability that it first appeared to provide, and it may serve conservation poorly through misdirected effort if it is used as the basis for setting policies or taking actions.     

Sample Size for the Diagnosis of Conservation Units

Abstract: Use of the phylogenetic species concept in defining conservation units is based on the assumption that the fixation of a particular character state in a population is diagnostic of a long history of reproductive isolation. In practice, diagnosis is usually based on the character states of a small sample of individuals rather than the states of the entire population. Unfortunately, when sample sizes are small, samples in which all individuals share one character state can easily be drawn from populations that are actually polymorphic. I describe statistical methods for examining how much confidence can be placed in the diagnosis of a conservation unit, given the operative sample size. The methods estimate the probability of drawing a sample in which all individuals show the same state, if individuals with unsampled ( hidden) states actually exist in the population at some hypothetical frequency (e.g., 0.05). I considered finite and infinite population-size models. The infinite population-size model suggests that in order to reject with 95% confidence the hypothesis that 5% of individuals carry hidden character states, a sample of 59 individuals is necessary. Finite population-size models give slightly smaller critical sample sizes for diagnosis with 95% confidence. I describe methods for including the effect of uncertainty in estimating population size when calculating critical sample size, and I discuss extensions to multiple characters and the impact of spatial structuring of character states. My results suggest that confident diagnosis requires sample sizes much larger than those commonly used when the phylogenetic species concept is applied to defining conservation units.